Prostacyclin (PGI2) formed by the vasculature is believed to be an essential and thrombagenic factor. Proteins are synthesized from arachidonic acid by the sequential actions of prostaglandin endoperoxide (PGH) synthase and PGI2 synthase. Little information is available concerning how PGI2 formation is regulated. We have prepared monoclonal antibodies against PGI2 synthase and have used these reagents to purify this enzyme for the first time. We now propose to determine its subunit composition, spectral properties, sensitivity to amino acid specific reagents and reactivity with inhibitory hydroperoxides. As an extension of these in vitro studies, we will determine if reinitiation of PGI2 synthesis in cultured bovine endothelial cells treated with hydroperoxides requires new protein synthesis and if hydroperoxide inhibition affects the rates of formation of either PGH or PGI2 synthase. The long range goal of these studies is to define what factors are important for optimizing vascular PGI2 synthesis. Preliminary immunocytofluorescence studies suggest that PGI2 synthase is located on the plasma membrane. We will resolve this question and determine if the enzyme exhibits basolateral-luminal asymmetry using Digital Fluorescence Image Processing after microinjection of endothelial cells with fluorescent-labeled anti-PGI2 synthase antibodies. These studies, besides defining the intracellular site of prostacyclin synthesis, will also indicate if there is vectoral release of PGI2 from the endothelium.